Gluconeogenesis in mammalian liver is regulated by both short-term and long-term controls. Long-term control is provided by induction and deinduction of key enzymes in the pathway, including P-enolpyruvate carboxykinase. Short-term controls also regulate P-enolpyruvate carboxykinase activity since sufficient catalytic capacity is present in deinduced rat liver to account for maximal observed rates of gluconeogenesis during the fasted or diabetic state. At present, the mechanism and regulation of P-enolpyruvate carboxykinase is not completely understood. The purpose of these studies will be to determine if regulation of the enzyme can occur, by a) product inhibition at physiological concentrations of Mg2 ions, GDP and P-enolpyruvate, b) a change in the reaction mechanism from P-enolpyruvate formation to pyruvate formation as the GTP/GDP ratio changes over physiological ranges and c) interaction of the enzyme with divalent transition metal ions. Studies of the mechanism will provide the true and apparent dissociation constants for substrates and products to be compare with cytosolic concentrations of these reactants. The mechanism of the partial reactions catalyzed by the enzyme will allow evaluation of the physiological significance of the oxalacetate decarboxylation reaction. Direct kinetic and binding studies with metal ions will allow evaluation of the role of Mg2 ions, Mn2 ions, Zn2 ions and Co2 ions in activating P-enolpyruvate carboxykinase.